The ultimate goal of this study is to identify, characterize, and determine the function of genes involved in critical steps in early vertebrate development. This proposal describes experiments that exploit recent advances in transgenic mouse technology as well as new methods for perturbing gene expression in chick embryos to study in detail the functions of two genes, Evx1 and Gbx2, which are known to play essential roles at early stages of vertebrate development. These methods will be used to perform both loss- and gain-of-function experiments to test hypotheses about Evx1 and Gbx2 function in the vertebrate embryo, including the idea that Evx1 functions to pattern the primitive streak and the idea that Gbx2 functions to pattern the embryonic brain via a repressive effect on Otx2 gene expression. In the course of these studies mouse lines will be produced that express the gene that encodes the site-specific DNA recombinase Cre in specific cell types. These lines, Hnf4-cre, T-cre, Sox1-cre, and Ap2alpha-cre, should be useful for specifically inactivating genes in the visceral endoderm, the nascent mesoderm and endoderm, the prospective neuroectoderm, and the distal limb mesenchyme, respectively. In addition to their utility in the experiments described here, these mouse lines should provide a valuable resource for studies aimed at analyzing the function of many other genes in the early embryo. By using them to elucidate the specific functions of Evx1 and Gbx2 insights should be gained into the fundamental mechanisms by which the embryonic ectoderm is prepared for gastrulation, the primitive streak is patterned to establish the basic body plan during gastrulation, and the neuroectoderm is patterned at the earliest stages in its development.